Progress 03/01/11 to 02/28/15
Outputs
Target Audience:Our target audience is researchers working on basic research in hormone signaling, as well as those trying to apply the understanding of the role of the phytohormone cytokinin in plant growth and development to applied areas of agriculture. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This grant provided support to train a postdoctoral fellow, Dr. Christian Burr, and an undergraduate studcent, Emily Jennings in various aspects of molecular genetic studies of rice. How have the results been disseminated to communities of interest?We have published our findings in several peer reviewed publications (noted above) and have presented our findings in multiple seminars to other departments (e.g. NCSU, U. Tennessee, Ohio U., McGill, U. of Missouri,Washington Uinversity, University of Nevada, UC Riverside,U. of Tennessee, Ohio University) and at multiple international conferences (21st IPGSA meeting, Shanghai China; Symposium on Auxins and Cytokinins in Plant Development, Prague; 1st CRC 1101 Symposium "Molecular Encoding of Specificity in Plant Processes" University of Tübingen). What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Cytokinins are N6-substituted adenine derivatives that play critical roles in plant growth and development, including cell division, apical dominance, sink-source regulation, grain filling, senescence, vascular development, meristem function, biotic and abiotic interactions, and stress tolerance. Cytokinin signaling has been well characterized in Arabidopsis and found to involve a canonical two-component pathway. We proposed to analyze two-component genes in rice, with a focus on their role in cytokinin signaling. Our first objective was to examine the expression of genes known to play a role in cytokinin function using a newly developed, highly sensitive method (NanoStrings) to quantify the level of multiple transcripts. The NanoStrings system is based on direct multiplexed measurement of gene expression using fluorescent "barcodes". We developed a NanoString probe set consisting of genes encoding proteins involved in cytokinin biosynthesis, metabolism and signaling. This aim has been met and the results published. In sum, the expression of these genes involved in cytokinin function was analyzed in rice roots and shoots and in response to phytohormones. The OsHK and OsAHP genes were all fairly highly expressed in both roots and shoots. In contrast, there was a wide variation in the relative expression of individual genes within the OsRR, LOG, OsIPT, and OsCKX gene families in both roots and shoots. Of the 66 genes involved in cytokinin function that were analyzed, 42 were present above background levels in roots and/or shoots. Many of the low abundant genes detected by NanoStrings were not detectable in other analyses, reflecting the high sensitivity of the NanoStrings assay. For example, of the eleven cytokinin oxidase genes, only OsCKX11 expression was reliably detected using Affymetrix GeneChip arrays, as compared to the eight OsCKX genes robustly detected using NanoStrings. Nearly all type-A OsRRs and OsHK4 were up-regulated in response to cytokinin, but other cytokinin signaling elements were not appreciably affected. We also examined the expression of this gene set to other phytohormones, including auxin, ABA, brassinosteroid and GA. We found differential effects of the hormones on the expression of these genes, with the biggest affects generally on the genes involved in cytokinin metabolism. Our next objective was to disrupt the function of cytokinin two-component signaling elements and then to assess the effect of these disruption on cytokinin responsiveness and the growth and development of rice. We focused on two classes of two-component elements: 1) the OsAHPs, which are presumably positive elements in cytokinin signaling and which are represented by only two functional genes; 2) a subset of type-A OsRR, which are presumable negative elements and which are encoded by a divergent gene family. To this end, we first focused on collecting T-DNA insertion alleles disrupting the OsAHP1 and OsHP2, and various type-A OsRR genes. We obtained and confirmed insertions for both of the OsAHPs and three of the thirteen type-A OsRRs. Subsequent to this initial work, the development of CRISPRs provided a much more powerful approach to reverse genetics. Thus, we developed constructs todisrupt both OsHP genes and the various type-A RRs. We were quite successful in this, and have developed lines disrupted for both OsHPs as well as single and multiple type-A RRs. These are an invaluable resource for the community to analyze the role of cytokinin in various processes. We characterized the effects of these loss-of-function mutations on the growth and development of rice, and on the response to cytokinin. We proposed to screen for proteins that interact specifically with the phosphorylated form of the type-A OsRRs. We have made a set of yeast two-hybrid bait clones expressing a fusion to several phosph-mimic type-A ARRs. Further, we have developed a high quality rice prey cDNA library from cytokinin treated seedlings and panicles. We have screened for interactors with activated OsRR2 and have found seven interacting proteins, including the type-A OsRR6 and an exocyst subunit. We also developed rice lines expressing tagged version of two type-A RRs to purify protein complexes from rice to identify additional interacting proteins in the future.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Schaller GE, Bishopp A, Kieber JJ. (2015) The yin-yang of hormones: cytokinin and auxin interactions in plant development. Plant Cell. 27(1):44-63
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Kieber JJ, Schaller GE. (2014) Cytokinins. The Arabidopsis Book. 12:e0168
- Type:
Journal Articles
Status:
Published
Year Published:
2012
Citation:
Tsai YC, Weir NR, Hill K, Zhang W, Kim HJ, Shiu SH, Schaller GE, Kieber JJ. (2012) Characterization of genes involved in cytokinin signaling and metabolism from rice. Plant Physiol. 158(4):1666-84.
- Type:
Journal Articles
Status:
Published
Year Published:
2012
Citation:
Kim HJ, Kieber JJ, Schaller GE. (2012) Overlapping and lineage-specific roles for the type-B response regulators of monocots and dicots. Plant Signal Behav. 7(9):1110-3
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